The objective of this research is the isolation, cloning, and characterization of genes that determine specific steps in the developmental pathway of the sexual cycle in the higher fungus, Schizophyllum commune. The pathway is initiated and regulated overall by a defined complex of mating-type genes, and expressions of the developmental genes to be studied are controlled by the mating-type genes. Representatives of three types of developmental genes, which have been well characterized by methods of classical genetics, have been chosen for study. The methods proposed involve the use of a newly developed DNA-mediated transformation system in Schizophyllum. Appropriate mutant and wild-type strains will be bred for recipient cells in which insertion of transfected developmental genes can be recognized by a defined alteration in phenotype. The donor DNA will consist of a mixed clone bank of Schizophyllum DNA in a vector containing a selective nutritional gene. It will be transfected into recipient cells with a matching auxotrophic mutation and a developmental phenotype appropriate for selection of the developmental gene to be isolated. Putative transformants for the nutritional gene will be selected first on the basis of prototrophy. These will be screened for an alteration in developmental phenotype indicative of cotransformation for the developmental gene. The putative cotransformants will be analyzed for stability of phenotype, meiotic segregation and the presence of integrated vector sequences. The gene of interest will then be rescued from stable transformants, by selection and cloning of vector sequence in E. coli, and will subsequently be tested for incorporation of the developmental gene by retransformation of Schizophyllum to the relevant phenotype in high frequency. Cloned sequences containing developmental genes will then be characterized by subcloning and sequencing. Ultimately, a comparison of sequences of several developmental genes should elucidate the molecular basis for control of their expressions by the trans-acting mating-type genes. (Comparable efforts to isolate and characterize the mating-type genes are in progress in other laboratories.) This system has been chosen as an accessible and intriguing model for the investigation of gene regulation of development, a phenomenon basic to the biology of other eukaryotes, including organisms important to human health.